Female specific pathogen-free (SPF) inbred BALB/c mice(6 weeks, 20–25 g)were obtained from Hunan SJA Laboratory Animal Co.,Ltd (Changsha, China; license #: SCXK (Xiang) 2013-0006. The animals were maintained in an SPF animal house, with a regular 12h/12 h light/dark cycle, relative humidity of 40–70%, and an average temperature of 25℃.
The study was carried out in compliance with the ARRIVE guidelines.The study was approved by the Animal Care and Use Committee of the Medical College of Jiangxi Medical College. Experiments were performed in accordance with the guidelines of the United States National Institutes of Health (NIH) regarding the care and use of animals for experimental procedures.
Chemicals and reagents.
OVA (CAS NO. 9006-59-1, Biotechnology grade), montelukast (CAS NO.151767-02-1, LC&T, purity ≥ 98%) and 18β-GA(CAS NO. 471-53-4, purity ≥ 98%) were obtained from Macklin Biochemical Co., Ltd. (Shanghai, China).
Asthmatic model induced by OVA and drug administration.
The mouse model of asthma was induced by intraperitoneal injection of OVA (20 µg), which was mixed with aluminum hydroxide (2 mg) on day 1 and day 14. From day 18 to day 23, the mice received oral gavage of Mon (10 mg/kg) and 18β-GA (10, 20, and 40 mg/kg). The control group received saline of the same amount. On day 21 to day 23, 1% OVA aerosol in PBS was given to the sensitized mice for 20 minutes. On day 24, whole-body plethysmography (Buxco Electronics, Troy, NY) was used to evaluate airway hyperresponsiveness35. There were 6 groups of mice, with 5 mice in each group (n = 5): Control (normal control), Asthma (OVA sensitization), Asthma + Mon (OVA sensitization and Mon administration), Asthma + 18β-GA 10, 20, and 40 (OVA sensitization and 18β-GA gavage of 10, 20, and 40 mg/kg, respectively). The procedure of the experiment is presented in Fig. 2.
Measurement of airway hyperresponsiveness to inhaled MCH.
The airway hyperresponsiveness was measured 24 h after the last OVA challenge. The experiment was carried out when the mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.): the mouse trachea was incised, then intubated and placed in the whole body plethysmograph, which was connected to the ventilator. Then, when the mice were exposed to increasing concentrations (0, 3.125, 6.25, and 12.5 mg/mL) of MCH, record the RL and Cdyn.
Lung tissues were fixed with 10% formalin, paraffin-embedded, and cut into 4 µm sections, then stained with H&E to assess infiltration of inflammatory cells, or stained with PAS to assess the mucus production of goblet cells. The inflammation of the lung tissue and mucus production was quantitatively analyzed by an image analyzer.
Assessment of bronchoalveolar lavage fluid and serum.
The mice were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and the lungs were exposed by thoracotomy. The mice were intubated and intratracheally instilled with PBS twice before the bronchoalveolar lavage fluid (BALF) collection. After these procedures were completed, the mice were sacrificed by cervical dislocation. The lavage solution was then centrifuged at 4°C and 500 g for 10 minutes. Differential cell counts were performed and stained with Diff-Quik (Solarbio, Beijing). The cytokines in the supernatant were detected and the cell precipitation was resuspended. The type and number of leukocytes were calculated by a cell counter. The levels of IL-5, IL-13, IFN-γ, TNF-α and IgE were determined by ELISA kits (Nanjing Jiancheng Bioengineering Institute, China).
Evaluation of lung oxidative stress.
Samples of lung tissues was cut up and homogenized with RIPA cold lysis buffer for 3 minutes. After centrifugation (12 000 g, at 4°C for 10 min), lung ROS, T-AOC, MDA, CAT, SOD and GSH-Px were measured on a 96 well plate by a commercially available kit (Beyotime Institute of Biotechnology). The activities of ROS, T-AOC, MDA, CAT, SOD, and GSH-Px were detected by the ROS, T-AOC, MDA, SOD, CAT, and GSH-Px assay kits, then the absorbance at 485 nm, 593 nm, 532 nm, 450 nm, 520 nm and 340 nm was measured by a microplate reader, respectively.
The lung tissue and lysate containing protease inhibitor were homogenized, then centrifuged at 4℃ at 12 000 g to retain the supernatant. The 25 µL of 15-fold diluted supernatant was used for protein quantification. The loading buffer was added, and the remaining supernatant was separated by SDS-PAGE, and then transferred to PVDF membrane. Then sealed at room temperature for 2 hours and incubated with the primary antibodies (1:1 000) overnight at 4 ℃. On the second day, the second antibody (1:15 000) was applied for 1 hour, and the ECL kit was used for photodetection. The primary antibodies including anti-HO-1 Abs, anti-NF-κB Abs, anti-phospho anti-Nrf2 Abs, and NF-κB Abs that were used in the study were purchased from Proteintech Technology Inc.
Data are expressed as mean ± standard deviation (SD). The analysis used one-way ANOVA with Tukey post hoc test. Graphs are generated using Graphpad prism 8. The statistically significances were set as the following: p < 0.05, p < 0.01 and p < 0.001.